Project Summary Hematopoiesis, the process by which hematopoietic stem and progenitor cells (HSPCs) in the bone marrow produce all mature blood cells for the hematopoietic system, is required for life. HSPCs are particularly prone to DNA replication stress due to their high rates of replication during normal hematopoiesis and certainly during bursts of proliferation from stress-induced/emergency hematopoiesis. DNA replication stress that is not properly resolved is postulated to contribute to genomic instability and cell death, ultimately leading to HSPC defects, deficiencies and/or bone marrow failure. Yet, the proteins that mitigate such stress in HSPCs remain largely uncharacterized. Understanding the proteins that respond to DNA replication stress in HSPCs is essential to understanding how HSPC development and function is protected throughout a lifetime. Recently, Smarcal1 and Zranb3, two proteins with the same biochemical activity of fork reversal and remodeling, were determined in human cancer cells to respond to DNA replication stress and prevent DNA replication fork collapse, but their role in vivo and in hematopoietic cells remained unexplored. The Eischen lab discovered that while Smarcal1 is not required for normal hematopoiesis, it is essential for HSPCs during stressed/emergency hematopoiesis. Currently, little is known about Zranb3 in vivo, as well as whether the functions of Smarcal1 and Zranb3 are redundant in vivo in hematopoietic cells. Our preliminary data suggest Zranb3 may be essential for HSPCs and its functions are non-redundant with Smarcal1 in vivo. Therefore, we hypothesize Zranb3 is an essential component of the DNA replication stress response in HSPCs and significantly contributes to their proliferation and survival. To test this hypothesis, we will use mouse models and both mouse and human primary hematopoietic cells. In Aim 1, we propose to investigate the requirements of Zranb3 to normal hematopoiesis and the mechanism involved. In Aim 2, we will investigate the contribution of Zranb3 to DNA replication stress- induced during stressed/emergency hematopoiesis. Results from the proposed experiments will significantly increase understanding of the DNA replication stress response that protects hematopoietic cells during normal and stressed hematopoiesis and prevents hematopoietic cell failure. The MD/PhD program at Thomas Jefferson University provides challenging, comprehensive training that will allow me to fulfill the goals of this application and become a successful independent physician-scientist. My training will include the development of many skills and an increase of knowledge through multiple approaches supported by my mentor, thesis committee, and other scientists. The educational and career development objectives outlined in this proposal will help me fulfill my goal of becoming a successful, independent physician- scientist focused on hematology.